Method To Develop High Oleic Acid Soybeans Using Conventional Soybean Breeding Techniques

ABSTRACT

The present invention is directed to a soybean plant with mutations in FAD2-1A and FAD2-1B. Moreover, the present invention is directed to seeds from said plants with altered ratios of monosaturated and polyunsaturated fats. In particular, the present invention is directed to plants where the plants exhibit elevated levels of oleic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/223,942 filed Jul. 8, 2009.

SEQUENCE LISTING

This application is accompanied by a sequence listing both on paper andin a computer readable form that accurately reproduces the sequencesdescribed herein.

BACKGROUND

Plant oils are used in a variety of applications. Novel vegetable oilcompositions and improved approaches to obtain oil compositions, frombiosynthetic or natural plant sources, are needed. Depending upon theintended oil use, various different fatty acid compositions are desired.Plants, especially species which synthesize large amounts of oils inseeds, are an important source of oils both for edible and industrialuses.

Oleic acid is a monounsaturated omega-9 fatty acid found in variousanimal and vegetable sources. It is considered one of the healthiersources of fat in the diet and is commonly used as a replacement for fatsources that are high in saturated fats.

Diets in which fat consumption are high in oleic acid have been shown toreduce overall levels of cholesterol, arteriosclerosis andcardiovascular disease. Specifically, oleic acid has been shown to raiselevels of high-density lipoproteins (HDLs) known as “good cholesterol”,while lowering low-density lipoproteins (LDLs) also known as the “bad”cholesterol. Thus, the development of new and inexpensive sources offoods comprising healthier forms of fatty acid is desirable.

Plants synthesize fatty acids via a common metabolic pathway known asthe fatty acid synthetase (FAS) pathway. Beta-ketoacyl-ACP (acyl carrierprotein moiety) synthases are important rate-limiting enzymes in the FASof plant cells and exist in several versions. Beta-ketoacyl-ACP synthaseI catalyzes chain elongation to palmitoyl-ACP (C16:0), whereasBeta-ketoacyl-ACP synthase II catalyzes chain elongation to stearoyl-ACP(C18:0). Beta-ketoacyl-ACP synthase IV is a variant of Beta-ketoacyl-ACPsynthase II, and can also catalyze chain elongation to 18:0-ACP. Insoybeans, the major products of FAS are 16:0-ACP and 18:0-ACP. Thedesaturation of 18:0-ACP to form 18:1-ACP is catalyzed by aplastid-localized soluble delta-9 desaturase (also referred to as“stearoyl-ACP desaturase”).

The products of the plastidial FAS and delta-9 desaturase, 16:0-ACP,18:0-ACP, and 18:1-ACP, are hydrolyzed by specific thioesterases (FAT).Plant thioesterases can be classified into two gene families based onsequence homology and substrate preference. The first family, FATA,includes long chain acyl-ACP thioesterases having activity primarily on18:1-ACP. Enzymes of the second family, FATB, commonly utilize 16:0-ACP(palmitoyl-ACP), 18:0-ACP (stearoyl-ACP), and 18:1-ACP (oleoyl-ACP).Such thioesterases have an important role in determining chain lengthduring de novo fatty acid biosynthesis in plants, and thus these enzymesare useful in the provision of various modifications of fatty acylcompositions, particularly with respect to the relative proportions ofvarious fatty acyl groups that are present in seed storage oils.

The products of the FATA and FATB reactions, the free fatty acids, leavethe plastids and are converted to their respective acyl-CoA esters.Acyl-CoAs are substrates for the lipid-biosynthesis pathway (KennedyPathway), which is located in the endoplasmic reticulum (ER). Thispathway is responsible for membrane lipid formation as well as thebiosynthesis of triacylglycerols, which constitute the seed oil. In theER there are additional membrane-bound desaturases, which can furtherdesaturate 18:1 to polyunsaturated fatty acids.

The soybean genome possesses two seed-specific isoforms of a delta-12desaturase FAD2, designated FAD2-1A and FAD2-1B, which differ at only 24amino acid residues. The genes encoding FAD2-1A and FAD2-1B aredesignated Glyma 10g42470 on Linkage Group 0 and Glyma 20g24530 onLinkage Group I on the soybean genome sequence, respectively (Glyma1.0,Soybean Genome Project, DoE Joint Genome Institute). FAD2-1A and FAD2-1Bare found in the ER where they can further desaturate oleic acid topolyunsaturated fatty acids. The delta-12 desaturase catalyzes theinsertion of a double bond into oleic acid (18:1), forming linoleic acid(18:2) which results in a consequent reduction of oleic acid levels. Adelta-15 desaturase (FADS) catalyzes the insertion of a double bond intolinoleic acid (18:2), forming linolenic acid (18:3).

TABLE 1 Characteristics of the major Fatty Acids Carbons:Double BondsName Saturation 16:0 Palmitic Acid Saturated 18:0 Stearic Acid Saturated18:1 Oleic Acid monounsaturated 18:2 Linoleic Acid ω-6 polyunsaturated18:3 α-Linolenic Acid ω-3 polyunsaturated

The designations (18:2), (18:1), (18:3), etc., refer to the number ofcarbon atoms in the fatty acid chain and the number of double bondstherein, Table 1. As used herein, the designations sometimes take theplace of the corresponding fatty acid common name. For example, oleicacid (18:1) contains 18 carbon atoms and 1 double bond, and is sometimesreferred to as simply “18:1”.

While previous research has demonstrated the important role of theFAD2-1A gene for increasing oleic acid, no reports have demonstrated adirect effect of the FAD2-1B gene on oleic acid accumulation. Soybean isa commodity crop that provides a major component of the fats and oils inthe American diet. Soybean is considered an oilseed, and it typicallycontains about 20% oleic acid as part of the fatty acid profile in theseed oil.

Soybean oil is used by the food industry in a variety of food productsincluding cooking oils, salad dressings, sandwich spreads, margarine,bread, mayonnaise, non-dairy coffee creamers and snack foods. Soybeanoil is also used in industrial markets such as biodiesel and biolubemarkets.

For many oil applications, low saturated fatty acid levels aredesirable. Saturated fatty acids have high melting points which areundesirable in many applications. When used as a feedstock or fuel,saturated fatty acids cause clouding at low temperatures, and conferpoor cold flow properties such as pour points and cold filter pluggingpoints to the fuel. Oil products containing low saturated fatty acidlevels may be preferred by consumers and the food industry because theyare perceived as healthier and/or may be labeled as “low in saturatedfat” in accordance with FDA guidelines. In addition, low saturate oilsreduce or eliminate the need to winterize the oil for food applicationssuch as salad oils. In biodiesel and lubricant applications, oils withlow saturated fatty acid levels confer improved cold flow properties anddo not cloud at low temperatures.

Various technologies for generating mid to high oleic acid levels insoybean plants are known. For example, U.S. Patent Publication No.2007/0214516 discloses a method for obtaining soybean plants that havemoderately increased levels of oleic acid. However, this technologyrequires the genetic modification of soybean plants through theintroduction of a transgene by transgenesis.

While transgenic soybean lines have been generated that produce soybeanoil containing mid to high levels of oleic acid, non-geneticallymodified (non-GMO) soybean plant lines that produce seed with mid tohigh oleic acid content is desirable.

SUMMARY

The presently disclosed instrumentalities overcome the problems outlinedabove and advance the art by providing a method to create and selectconventional non-GMO soybean lines containing greater than around 20%and up to around 85% oleic acid in soybean seed oil with up to afour-fold increase over the levels produced by commodity soybeans. Theinstrumentalities described herein, demonstrate the ability toefficiently incorporate an enhanced oil quality trait into elitevarieties of soybean plants without the expensive testing and evaluationused in traditional soybean breeding.

The presently disclosed instrumentalities demonstrate that mutation inthe FAD2-1B gene alone resulted in very minor increases in oleic acidlevels. However, combinations of mutations in the FAD2-1A and FAD2-1Bgenes resulted in dramatic increases in oleic acid level of the seedoil.

In an embodiment, a soybean plant having one or more mutations in theFAD2-1A and FAD2-1B genes, wherein seed from said plant has about 75% toabout 85% oleic acid content

In an embodiment a soybean plant expressing a mutated FAD2-1B geneencoded by a polynucleotide having at least 70%, 80%, 90%, 95%, 98%, or99% identity with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 andexpressing a mutated FAD2-1A gene encoded by a polynucleotide having atleast 70%, 80%, 90%, 95%, 98%, or 99% identity with the sequence of SEQID NO: 3 or SEQ ID NO: 4 has seed with a modified fatty acid compositionthat is about 75% to about 85% oleic acid.

In an embodiment, a method of selecting soybean plants with seed havingan oleic acid content of between about 65% to about 85%, said methodcomprising: crossing a first soybean plant having one or more mutationsin a first polynucleotide sequence encoding a FAD2-1A gene SEQ ID NO: 5with a second soybean plant having one or more mutations in a secondpolynucleotide sequence encoding a FAD2-1B gene SEQ ID NO: 6 isdescribed.

In an embodiment, a nucleic acid encoding for expression of a mutatedform of FAD2-1B, comprising: a sequence length of at least 72nucleotides (24 amino acids) encoding for the expression of SEQ ID NO: 6wherein the sequence includes at least one mutation selected from thegroup consisting of: a non-conserved amino acid substitution at aminoacid position 137, b. a non-conserved amino acid substitution at aminoacid position 143 is described.

In an embodiment, a soybean plant expressing a mutated FAD2-1B geneencoded by a polynucleotide having at least 70%, 80%, 90%, 95%, 98%, or99% identity with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 has seedwith a modified fatty acid composition that is about 22% to about 41%oleic acid.

In an embodiment, a soybean plant expressing a mutated FAD2-1B gene thatresults in a reduced activity of the FAD2-1B has seed with a modifiedfatty acid composition of oleic acid levels greater than about 20%.

In an embodiment, a transgenic soybean plant expressing a dominantnegative form of FAD2-1B has seed with a modified fatty acid compositionof oleic acid levels greater than 20% preferably between about 20% to60% and most preferably between about 60% to 85%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are weblogo outputs showing amino acid conservation offatty acid desaturase enzymes.

FIG. 2 is a bar graph illustrating the relative fatty acid levels as afunction of total fatty acids of progeny from M23×PI 283327 recombinantinbred lines.

FIG. 3 is a bar graph illustrating the oleic acid content as function oftotal fatty acids of parents and progeny from M23×PI 283327 recombinantinbred lines.

FIG. 4 is a bar graph illustrating the oleic acid content as function oftotal fatty acids of progeny from 17D×PI 283327 F2 seeds.

FIG. 5 is a bar graph illustrating oleic acid levels as a function oftotal fatty acids of progeny from M23×PI 567189A recombinant inbredlines.

FIG. 6 is a bar graph illustrating oleic acid levels as a function oftotal fatty acids of progeny from Jake×PI 283327 recombinant inbredlines.

FIG. 7 is a graphical representation of a melting curve analysis used todetermine genotype of various FAD2 alleles.

FIG. 8 is a bar graph illustrating oleic acid levels as a function oftotal fatty acids for population 1.

FIG. 9 is a bar graph illustrating oleic acid levels as a function oftotal fatty acids for population 2.

FIG. 10 is a bar graph illustrating oleic acid levels as a function oftotal fatty acids for population 3.

DETAILED DESCRIPTION

As used herein, “allele” refers to any of one or more alternative formsof a gene locus, all of which alleles relate to a trait orcharacteristic. In a diploid cell or organism, the two alleles of agiven gene occupy corresponding loci on a pair of homologouschromosomes.

As used herein, “FAD2” refers to a gene or encoded protein capable ofcatalyzing the insertion of a double bond into a fatty acyl moiety atthe twelfth position counted from the carboxyl terminus. FAD2 proteinsare also referred to as “delta-12 desaturase” or “omega-6 desaturase”.The term “FAD2-1A” is used to refer to a FAD2 gene or protein defined asGlyma10g42470.1 in the Glyma1.0 whole genome sequence(http://www.phytozome.net/soybean) that is naturally expressed in aspecific manner in seed tissue, and the term “FAD2-1B” is used to refera FAD2 gene or protein defined as Glyma20g24530.1 in the Glyma1.0 wholegenome sequence (http://www.phytozome.net/soybean) that is (a) adifferent gene from a FAD2-1A gene or protein and (b) is naturallyexpressed in multiple tissues, including the seed.

As used herein, “gene” refers to a nucleic acid sequence thatencompasses a 5′ promoter region associated with the expression of thegene product, any intron and exon regions and 3′ or 5′ untranslatedregions associated with the expression of the gene product.

As used herein, “genotype” refers to the genetic constitution of a cellor organism.

As used herein, “phenotype” refers to the detectable characteristics ofa cell or organism, which characteristics are the manifestation of geneexpression

As used herein, non-genetically modified (non-GMO) means reasonablycapable of occurring in nature. An organism is considered non-GMO if ithas not been genetically engineered through the addition of exogenous,or recombinant nucleic acid, such as a transgene, to alter the geneticconstitution of the organism.

As used herein, “crossing”, as used herein, refers to the mating of twoparent plants.

As used herein, “F1” refers to first generation progeny of the cross oftwo plants.

As used herein, “F2” refers to second generation progeny of the cross oftwo plants.

As used herein, “F3”, as used herein, refers to third generation progenyof the cross of two plants.

As used herein, “F4”, as used herein, refers to fourth generationprogeny of the cross of two plants.

As used herein, “F5”, as used herein, refers to fifth generation progenyof the cross of two plants.

As used herein, “F6”, as used herein, refers to sixth generation progenyof the cross of two plants.

As used herein, “F7”, as used herein, refers to seventh generationprogeny of the cross of two plants.

As used herein, “F8”, as used herein, refers to eighth generationprogeny of the cross of two plants.

As used herein, a recombinant inbred line (RIL) is produced to form apermanent and stable quantitative trait locus (QTL) mapping resource. Inthe first step of the development of RILs, two parental inbred lines arecrossed (mated) together to form a uniformly heterozygous F1 generation.The F1 are intermated (or selfed) to form an F2 generation; mostindividuals in the F2 will contain recombinant chromosomes resultingfrom crossovers between the two purely parental chromosomes present ineach F1 plant. The parental alleles are said to be segregating in the F2generation, since it is a matter of chance just which of the threecombinations of parental alleles will occur in a given F2 plant.Numerous individuals from the segregating F2 generation then serve asthe founders of corresponding RILs. Each subsequent generation of agiven RIL is formed by selfing in the previous generation and withsingle seed descent. In this manner each RIL, after several generations,will contain two identical copies of each chromosome, with most of thembeing recombinant. Each individual RIL will contain a different mix ofrecombinant and parental chromosomes, with a unique set of recombinationbreakpoint locations across the genome. Taken as a group, the set ofRILs form a segregant QTL mapping population which can be stablyregenerated year after year via single seed descent.

As used herein genotypic designations are as follows:

AABB—homozygous wild-type FAD2-1A and homozygous wild-type FAD2-1B;aaBB—homozygous mutant FAD2-1A (mFAD2-1A) and homozygous wild-typeFAD2-1B;AAbb—homozygous wild-type FAD2-1A and homozygous mutant FAD2-1B(mFAD2-1B);aabb—homozygous mFAD2-1A and homozygous mFAD2-1B

As used herein, the soybean plant lines designated “Jake” and “Williams82” (W82) are conventional soybean varieties that have wild-type levelsof oleic acid and wild-type alleles of FAD2-1A and FAD2-1B.

As used herein a Plant Introduction (PI) or plant introduction line is asoybean line assumed to be inbred for multiple generations so that itsprogeny stably inherit all of the genes that it contains. Plantintroduction lines can be local landraces, cultivars, varieties, fieldcollections of locally adapted lines, selections from any of theselines, or advanced breeding lines that have been inbred and havestabilized genomes. The National Plant Germplasm System maintains acollection of Glycine max lines referred to as Plant Introductions.

As used herein, a maturity group is an agreed-on industry division ofgroups of varieties based on zones in which they are adapted, primarilyaccording to day length or latitude. They consist of very long daylength varieties (Groups 000, 00, 0), and extend to very short daylength varieties (Groups VII, VIII, IX, X).

A “fatty acid” is a carboxylic acid that generally has a long unbranchedaliphatic carbon chain. The designations (18:2), (18:1), (18:3), etc.,refer to the number of carbon atoms in the fatty acid chain and thenumber of double bonds therein, respectively. For example, oleic acid(18:1) contains 18 carbon atoms and 1 double bond. Exemplary fatty acidsinclude:

-   -   omega-3 fatty acids such as:        -   alpha-linolenic acid (CH₃(CH₂CH═CH)₃(CH₂)₇COOH)    -   omega-6 fatty acids such as:        -   linoleic acid (CH₃(CH₂)₄—CH═CHCH₂CH═CH(CH₂)₇COOH)    -   omega-9 fatty acids such as:        -   oleic acid (CH₃(CH₂)₇CH═CH(CH₂)₇COOH)    -   and saturated fatty acids such as:        -   palmitic acid (CH₃(CH₂)₁₄COOH)        -   stearic acid (CH₃(CH₂)₈COOH).

An isolated nucleic acid, as used herein, means a nucleic acid that isfree of at least some of the contaminants associated with the nucleicacid or polypeptides occurring in a natural environment and that has asequence that can encode for a gene.

An isolated nucleic acid can be further defined as among other things, afragment or a part of the nucleic acid, such as a short sequence ofbases from the nucleic acid of at least a length claimed, or a nucleicacid encoding for a truncated form, a modified form, or an isoform ofthe protein or polypeptide encoded by the nucleic acid. An isolatednucleic acid may include DNA from which the introns are removed. Anisolated nucleic acid may be under the control of an exogenous promoter.

As used herein, a mutation may be one or more nucleotide deletions,substitutions or insertions in a polynucleotide sequence. A mutation maybe one or more of a missense, nonsense, frameshift, insertion ordeletion.

As used herien, a missense mutation is a point mutation in which asingle nucleotide is changed in a gene sequence, resulting in an aminoacid change in the corresponding amino acid. A missense mutation mayresult in reduced activity of the protein encoded by the gene, or mayresult in a nonfunctional protein.

As used herein, a nonsense mutation is a mutation in a sequence of DNAthat results in a premature stop codon, or a nonsense codon in thetranscribed mRNA, and may result in a truncated protein product.Nonsense mutations may result in reduced activity of the protein encodedby the gene, or may result in a nonfunctional protein.

As used herein, a frameshift mutation is a genetic mutation in apolynucleotide sequence caused by insertion or deletion of a number ofnucleotides that is not evenly divisible by three. Due to the tripletnature of gene expression by codons, the insertion or deletion candisrupt the reading frame, or the grouping of the codons, resulting in adifferent translated protein product than from the original non mutatedgene. Frameshift mutations may result in reduced activity of the proteinencoded by the gene, or may result in a nonfunctional protein.

As used herein, a deletion results in the loss of any number ofnucleotides e.g. from a single base to an entire gene and surroundingpolynucleotide sequences. A deletion mutation may result in reducedactivity of the protein encoded by the gene, or may result in anonfunctional protein.

As used herein, an insertion results in the addition of any number ofnucleotides e.g. from a single base to many thousands of bases. Aninsertion mutation may result in reduced activity of the protein encodedby the gene, or may result in a nonfunctional protein.

As used herein, a loss of function mutation is a mutation that renders aprotein incapable of carrying out its biological function.

Mutations in isolated polynucleic acids may be made by techniques knownin the art such as, but not limited to, site directed mutagenesis.

Mutations may be induced by X-ray, gamma ray or fast neutronirradiation, and treatment with chemical mutagens such as the alkylatingagents ethyl-methanesulfonate (EMS) or N-nitroso-N-methylurea NMU). Inaddition, natural genetic variation can result from mutations that arisefrom random DNA polymerase errors that occur during DNA replication of aplant genome. Natural genetic variation in plants may also result fromactivation of DNA repair mechanisms after exposure to natural sources ofionizing or nonionizing radiation.

Soybean plants can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in soybeans either by selfpollination or natural cross pollination, which typically is aided bypollinating organisms. In either natural or artificial crosses,flowering and flowering time are an important consideration. Soybean isa short-day plant, but there is considerable genetic variation forsensitivity to photoperiod. The critical day length for flowering rangesfrom about 13 h for genotypes adapted to tropical latitudes to 24 h forphotoperiod-insensitive genotypes grown at higher latitudes. Soybeansseem to be insensitive to day length for 9 days after emergence.Photoperiods shorter than the critical day length are required for 7 to26 days to complete flower induction.

Soybean flowers typically are self-pollinated on the day the corollaopens. The stigma is receptive to pollen about 1 day before anthesis andremains receptive for 2 days after anthesis, if the flower petals arenot removed. Filaments of nine stamens are fused, and the one nearestthe standard is free. The stamens form a ring below the stigma untilabout 1 day before anthesis, then their filaments begin to elongaterapidly and elevate the anthers around the stigma. The anthers dehisceon the day of anthesis, pollen grains fall on the stigma, and within 10h the pollen tubes reach the ovary and fertilization is completed.Self-pollination occurs naturally in soybean with no manipulation of theflowers. For the crossing of two soybean plants, it is typicallypreferable, although not required, to utilize artificial hybridization.In artificial hybridization, the flower used as a female in a cross ismanually cross pollinated prior to maturation of pollen from the flower,thereby preventing self fertilization, or alternatively, the male partsof the flower are emasculated using a technique known in the art.Techniques for emasculating the male parts of a soybean flower include,for example, physical removal of the male parts, use of a genetic factorconferring male sterility, and application of a chemical gametocide tothe male parts.

Either with or without emasculation of the female flower, handpollination can be carried out by removing the stamens and pistil with aforceps from a flower of the male parent and gently brushing the anthersagainst the stigma of the female flower. Access to the stamens can beachieved by removing the front sepal and keel petals, or piercing thekeel with closed forceps and allowing them to open to push the petalsaway. Brushing the anthers on the stigma causes them to rupture, and thehighest percentage of successful crosses is obtained when pollen isclearly visible on the stigma. Pollen shed can be checked by tapping theanthers before brushing the stigma. Several male flowers may have to beused to obtain suitable pollen shed when conditions are unfavorable, orthe same male may be used to pollinate several flowers with good pollenshed.

The plants of the present invention may be used in whole or in part.Preferred plant parts include reproductive or storage parts. The term“plant parts” as used herein includes, without limitation, seed,endosperm, ovule, pollen, roots, tubers, stems, leaves, stalks, fruit,berries, nuts, bark, pods, seeds and flowers. In an embodiment of thepresent invention, the plant part is a seed.

In one aspect, an isolated polynucleotide may comprise the nucleotidesequence of the PI 283327 mFAD2-1B (SEQ ID NO: 1) or fragment thereof.Alternatively, a polynucleotide may have substantial sequence similarityto SEQ ID NO: 1, for example, with at least 80%, 90%, 95%, 98%, or 99%sequence identity to the sequence of SEQ ID NO: 1 In another aspect, apolynucleotide may have substantial sequence similarity to thenucleotide sequence of PI 567189A mFAD2-1B (SEQ ID NO: 2), for example,with at least 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to thesequence of SEQ ID NO: 2.

The expression of a protein is generally regulated by a non-codingregion of a gene termed a promoter. When a promoter controls thetranscription of a gene, it can also be said that the expression of thegene (or the encoded protein) is driven by the promoter. When a promoteris placed in proximity of a coding sequence, such that transcription ofthe coding sequence is under control of the promoter, it can be saidthat the coding sequence is operably linked to the promoter. A promoterthat is not normally associated with a gene is called a heterologouspromoter.

In an embodiment the expression of the delta-12 desaturase proteinencoded by either SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3 and orSEQ ID NO: 4, alone or in combination may function as a, “dominantnegative” protein mutation. Dominant negative, or antimorphic mutationsoccur when the gene product adversely affects the normal, wild-type geneproduct within the same cell. This usually occurs if the product canstill interact with the same elements as the wild-type product, butblock some aspect of its function. Such proteins may be competitiveinhibitors of the normal protein functions.

The peptides encoded by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, andSEQ ID NO: 4 of the present disclosure may be prepared by chemicalsynthesis known to those of skill in the art. The peptides may also beproduced using an expression vector having a nucleotide sequenceencoding the peptide(s) of choice. The nucleotide sequence may beoperably linked to an appropriate promoter, enhancer, terminator, orother sequences capable of regulating the expression of the encodedpeptide. The nucleotide sequence may also be operably linked to otherfunctional sequences. In one aspect, such a functional sequence may be asequence encoding a purification tag, to facilitate expression andpurification of the peptides. In another aspect, such a functionalsequence may encode an accessory peptide that confers upon the corepeptide various properties that are beneficial for the therapeuticfunctionality of the core peptide, for example, by increasing thestability of the core peptide, or by facilitating the delivery of thecore peptide to its therapeutic target tissue or organ in the body.

The terms “protein,” “polypeptide,” “peptide,” and “enzyme” may be usedinterchangeably in this disclosure, all of which refer to polymers ofamino acids. In addition to the peptides explicitly disclosed herein,certain “conservative” substitutions may be made on these peptideswithout substantially altering the functionality of the peptides.

As generally understood in the art, conserved amino acid residues amongorthololgous proteins are the result of evolutionary pressure tomaintain biological function and/or folding the protein. An amino acidposition conserved among orthologous sets of genes can be involved inmany aspects of structure and function. Invariant positions, or thoseshowing conservation of certain residue properties (e.g. charge,hydrophobicity, etc.) are less likely to tolerate mutations than thosewhere the protein family permits mutations to a great variety of aminoacids. Positional amino acid sequence conservation based on databasesequence deposits, for example, is useful in the determination of aminoacid substitutions that may have a deleterious affect on protein foldingand/or biological function.

Computer algorithmic sequence alignment programs may be used to predictwhether an amino acid substitution affects protein function based onsequence homology and the physical properties of amino acids. Amino acidsubstitution prediction methods such as, but not limited to, SIFT,PolyPhen, SNPs3D, PANTHER PSEC, PMUT and TopoSNP may be used to predictthe effect of an amino acid substitution on protein function. Suchprediction methods may be used to determine amino acid substitutionsthat may result in a loss of function or a reduced activity of theFAD2-1A and/or FAD2-1B genes.

Conservative amino acid substitutions are generally defined as thereplacement of one or more amino acids for a different amino acid oramino acids, that preserve the structural and functional properties ofproteins.

“Non-conservative” substitutions of one amino acid for another aresubstitutions of amino acids having dissimilar structural and/orchemical properties, and are generally based on differences in polarity,charge, hydrophobicity, hydrophilicity and/or the amphipathic nature ofthe residues involved. The substituting amino acids may includenaturally occurring amino acids as well as those amino acids that arenot normally present in proteins that exist in nature.

The following examples illustrate the present invention. These examplesare provided for purposes of illustration only and are not intended tobe limiting. The chemicals and other ingredients are presented astypical components or reactants, and various modifications may bederived in view of the foregoing disclosure within the scope of theinvention.

Example 1 Isolation and Characterization of High Oleic Acid ContentSoybean Plant Lines

About 40 soybean strains with elevated oleic acid content were selected.Three breeding lines, including a patented accession strain M23 (U.S.Pat. No. 7,326,547), were noted as having different genes that affectoleic acid concentration. M23 has an oleic acid content of about 40%-50%of its total fatty acid profile. As described below, fatty acid profilesare represented as a percent of total seed fatty acid content. M23 has asingle recessive gene, designated as ol for higher oleic acid content(Takagi, Y. & Rahman, S. M Inheritance of high oleic acid content in theseed oil) of soybean mutant M23. Theoretical Applied Genetics 92,179-182 (1996)). A recent study revealed that ol in M23 is the result ofa deletion at the FAD2-1A locus (Sandhu et al., 2007). The other twobreeding lines were plant introductions (PI) with elevated oleic acidcontent based on fatty acid data from the Germplasm ResourcesInformation Network (GRIN). GRIN showed that strains PI 283327 and PI567189A each contained about 41% and 38% oleic acid content,respectively. However, in the University of Missouri-Delta CenterPortageville Mo. field tests across six environments between 2005-2007,strains PI 283327 and PI 567189A averaged about 30% oleic acid where asa check cultivar commonly grown by farmers averaged about 22% oleic acidcontent. These two PIs were later discovered to have mutations at theFAD2-1B locus which results in the higher seed oleic acid content.

Selection and Crosses

Recombinant inbred line from (RIL) population 1 (F6 RIL of Jake×PI283327), 2 (F2:6 and F2:7 RIL of M23×PI283327) and 3 (F2:5 and F2:7 RILof M23×PI 567189 A) were created at the same time. Three crosses weremade in summer 2005 at the Delta Research Center at Portageville, Mo.including Jake×PI 283327, M23×PI 283327 and M23×PI 567189A. PI 283327and PI 567189A are two elevated oleic acid lines with maturity group Vand IV, respectively (GRIN USDA), while Jake is a conventional highyielding soybean in group V that contains a typical oleic acid content(Shannon, J. G. et al. Registration of ‘Jake’ Soybean. Journal of PlantRegistration 129-30 (2007)). M23 was selected for elevated oleic acidafter mutagenesis of the cultivar Bay (Takagi, Y. & Rahman, S. M.Inheritance of high oleic acid content in the seed oil) of soybeanmutant M23. Theoretical Applied Genetics 92, 179-182 (1996). In 2005 andearly 2006, F1 seeds were advanced to the F2 generation in Costa Rica.Each RIL tracing to a single F2 plant except population 1 was alsoadvanced in Costa Rica from 2006 to 2007 for F5 seeds. In 2007, a bulkof five seeds from each RIL in each population was analyzed to obtainfatty acid profile for the Costa Rica location. Population 1 was grownin Portageville, Mo. to produce F7 seeds. Population 2 was grown inPortageville, Mo. to produce F6 seeds, and then soybean RILs with morethan 60% oleic acid were advanced to the F7 generation. In population 3,only F5 RILs producing more than 60% oleic acid were selected togenerate F7 seeds at Portageville, Mo. in subsequent generations.

In the paragraph immediately above, the nomenclature F2:6 meansF2-derived F6, meaning that the last common ancestor of the lines was atF1. The F2 plants started the single seed descent to the F6 generation.A representative sample of population 2 constituting at least 2500 seedshas been placed in a deposit according to terms of the Budapest Treatyfor conditional release upon of the seeds the granting of an issuedpatent. This deposit is designated PTA 11061.

In 2008, populations 1 and 2 were grown in Portageville, Mo. to producethe seeds analyzed for fatty acids in FIGS. 8 and 9. Data in FIG. 10 wasfrom F5 seeds of population 3 produced in Costa Rica. In addition, fivelines with the highest oleic acid content from populations 2 and 3 weregrown in Columbia, Mo. in 2009. In 2009, population 4 (17D×(PI283327×Jake)] was grown in Columbia, Mo. to produce the seeds analyzedfor fatty acid analysis in FIG. 5. Similarly, four to eleven lines fromeach of four combinations of homozygous FAD2-1A and FAD2-1B genes frompopulation 4 were grown in Columbia Mo. and selected lines frompopulation 4 were grown in Portageville, Mo. in 2009.

Population 5 was initiated in summer 2008 at Portageville, Mo. Soybeanline KB07-1#123 was crossed with soybean line #93 from population 2.Soybean line #93 (>80% oleic acid) was genotyped to contain the FAD2-1AΔ alleles from M23 and the FAD2-1B P137R alleles derived from PI 283327.KB07-1#123 is a soybean line with the pedigree [W82×(M23×10-73)]. Thissoybean line was selected to contain three mutant alleles affecting thefatty acid profile, including FAD2-1A Δ alleles from M23, and mutantFAD3A and FAD3C alleles from soybean line 10-73 (Dierking, E. & Bilyeu,K. New sources of soybean seed meal and oil composition traitsidentified through TILLING. BMC Plant Biology 9, 89 (2009); Bilyeu, K.,Palavalli, L., Sleper, D. & Beuselinck, P. Mutations in soybeanmicrosomal omega-3 fatty acid desaturase genes reduce linolenic acidconcentration in soybean seeds. Crop Science 45, 1830-1836 (2005). F1seeds were genotyped to confirm the heterozygosity and then advanced toobtain F2 seeds in summer 2009 at Bradford Research and ExtensionCenter, Columbia Mo.

Selection for desirable traits may occur at any segregating generation(F2 and above). Selection pressure may be exerted on a population bygrowing the population in an environment where the desired trait ismaximally expressed and the individuals or lines possessing the traitcan be identified. For instance, selection can occur for diseaseresistance when the plants or lines are grown in natural orartificially-induced disease environments, and the breeder selects onlythose individuals having little or no disease and are thus assumed to beresistant.

Double mutant, i.e. mFAD2-1A and mFAD2-1B, soybean plant lines may varyin oleic acid concentration depending on the environment, however theoleic acid content (generally up to around 80%-85% oleic acid content)is consistently higher than either wild type or single mFAD1A ormFAD2-1B mutant soybean plant lines.

Crossing of M23 and either PI 283327 or PI 567189A resulted in progenywith levels of oleic acid (around 85% and around 65% respectively) thatare significantly higher than either parent (around 20%-50%). This islikely the result of the combination of mutated alleles of FAD2-1Aderived from M23, and FAD2-1B derived from PI 283327 or PI 567189A.

When combining a different FAD2-1A gene, from strain 17D (17D has mutantFAD2-1A S117N allele and 35% oleic acid, developed by mutagenesis ofWilliams 82 seed)×PI 283327, 80% oleic acid lines were also identified.Regardless of the source of the two genes, inheritance of both mutatedFAD2-1A and FAD2-1B genes into a single genotype resulted in at leasttwice the oleic concentration than either parent.

Genetic Characterization of FAD2-1A and FAD2-1B Mutations

For initial characterization of the FAD2-1A and FAD2-1B alleles frommultiple germplasm lines, the FAD2-1A and FAD2-1B genes were amplifiedby PCR and sequenced. Genomic DNA was isolated from approximately 30 mgground seed using the DNeasy Plant Mini Kit (Qiagen, Inc., Valencia,Calif.). 5 to 50 ng of genomic DNA was used per PCR reaction. PCR wascarried out using Ex Taq according to manufacturer's recommendation(Takara, Otsu, Shiga, Japan) in a PTC-200 thermocycler (MJResearch/Bio-Rad, Hercules, Calif.). The forward primer for FAD2-1A was5′-ACTGCATCGAATAATACAAGCC-3′(SEQ ID NO: 7); and reverse primer was5′-TGATATTGTCCCGTGCAGC-3′(SEQ ID NO: 8); The forward primer for FAD2-1Bwas 5′-CCCGCTGTCCCTTTTAAACT-3′(SEQ ID NO: 9); and reverse primer was5′-TTACATTATAGCCATGGATCGCTAC-3′(SEQ ID NO: 10);. PCR conditions were:95° C. for 5 minutes followed by 34 cycles of 95° C. for 30 seconds, 60°C. for 30 seconds, 72° C. for 1 minute 30 seconds. PCR products wereexamined for size by running on Flashgel for 5 minutes. PCR productswere then isolated with the Qiaprep Spin Miniprep kit (Qiagen, Inc.) andsequenced at the University of Missouri DNA core facility using theforward and reverse primers for both FAD2-1A and FAD2-1B. Sequence datawas compared with reference “wild-type” Williams 82 sequence (W 82) forthe FAD2-1A and FAD2-1B genes. Comparative sequence analysis of alllines tested is illustrated in Table 2.

As illustrated in Table 2, “S>F” represents a serine to phenylalanineamino acid substitution. “M>V” represents a methionine to valine aminoacid substitution. “P>R” represents a proline to arginine amino acidsubstitution. “I>T” represents an isoleucine to threonine amino acidsubstitution.

TABLE 2 Variants in DNA sequences of FAD2-1B mutants Nucleotide 257 376410 428 724 Position 66 105 (S > F) (M > V) (P > R)  (I > T) 636657/669/682 (M > L) 918 Soybean lines W 82 G A C A C T C CTT T API437593 B, PI467310, PI404160B, G TCC G PI561338A, PI561315, PI603452PI567155 B T G TCC G PI592974, PI196165, G G G PI416908, PI458044PI578451, PI567189A G T G C TCC G PI210179, PI283327 G T G G TCC GPI567205 A PI458238 A G G G PI506885, PI507307 A T G TCC G PI507420 A GG TCC G

DNA sequence analysis revealed that PI 283327 was found to contain a Cto G nucleotide substitution at nucleotide 410 in the coding sequence(mRNA) of FAD2-1B resulting in a proline to arginine amino acidsubstitution missense mutation at amino acid 137 (P137R). In contrast,PI 567189A was found to contain a T to C nucleotide substitution atnucleotide 428 in the coding sequence of FAD2-1B resulting in anisoleucine to threonine missense mutation at amino acid 143 (I143T).Other single nucleotide polymorphisms were present in the allele, buteither did not change the amino acid sequence (silent mutations),contained missense mutations substituting similar amino acids(methionine to valine at amino acid position 125 (M125V), for example),or missense mutations in nonconserved regions of the protein (serine tophenylalanine at amino acid position 86 (S86F), for example).

Previously, investigation of the S86F mutation in a different germplasmaccession with this mutation, was not associated with an increase inoleic acid content, even in the presence of the FAD2-1A deleted allelefrom M23. The FAD2-1B P137R mutation is in a very conserved position inthe protein, while the I143T mutation is in a less conserved position(FIG. 1B). Subsequent to these discoveries, PI 210179 was found tocontain a FAD2-1B allele identical to PI 283327. PI 578451 was found tocontain a FAD2-1B allele identical to PI 567189A. Other germplasmaccessions containing variant FAD2-1A and FAD2-1B alleles were alsodiscovered by sequencing.

FIG. 1B shows the relative frequency of amino acid substitutions betweenamino acids 135-150 of the FAD2 gene sequences present in the NationalCenter for Biotechnology Information sequence database. A Weblogo outputwas determined by the amino acid conservation of fatty acid desaturaseenzymes aligned as part of the BLINK feature at NCBI using GI number197111724. Amino acid positions within the protein are listed on the Xaxis. The overall height for each amino acid column stack indicates thesequence conservation at that position while the height of one-letteramino acid symbols within the column stack indicates the relativefrequency of each amino acid in that position [Crooks G E, Hon G,Chandonia J M, Brenner S E WebLogo: A sequence logo generator, GenomeResearch, 14:1188-1190, (2004)]. The white and black arrows indicate theP137R and I143T positions mutated in PI 283327 and PI 567189A,respectively.

FIG. 1A is reproduced from Dierking and Bilyeu, 2009, BMC Plant Biology9:89 to show Weblogo output of the relative frequency of amino acidsubstitutions/amino acid conservation between amino acids 104-123 of theFAD2 gene. Amino acid positions within the protein are listed on the Xaxis. The overall height for each amino acid column stack indicates thesequence conservation at that position while the height of one-letteramino acid symbols within the column stack indicates the relativefrequency of each amino acid in that position. The arrow indicates theFAD2-1AS117N position mutated in line 17D.

Much work has been done with the M23 FAD2-1A gene, but initial resultswith the 17D line suggest that 80% oleic acid soybean lines can beproduced with either source of the FAD2-1A mutation in combination witha FAD2-1B mutation (described below).

The High Oleic Acid Phenotype is Stable in Plants Grown in AlternateEnvironments

Some of the high oleic acid soybean lines developed in this studydemonstrated stability for the high oleic acid trait when grown indifferent environments (Table 3). Of the three environments, Costa Ricatypically has the warmest temperatures during seed development, followedby the Portageville, Mo. environment; the Columbia, Mo. environment isthe coolest of the three environments during seed development. Thedifferences in the oleic acid contents between environments when theFAD2-1B P137R alleles were present were minor. Soybean lines withgenotype aabb of population 2 and 4 produced more than 80% oleic acidcontent in Costa Rica and Portageville, Mo. environments, and the oleicacid level was an average of 2-4% lower when grown in the Columbia, Mo.environment. It is notable that the variation in the phenotype wasnarrow in all of the environments. In contrast, the aabb soybean linesof population 3 containing the FAD2-1B I143T alleles had lower and morevariable oleic acid content in the cooler environments, and failed toproduce a high oleic acid phenotype in either the Columbia, Mo. orPortageville, Mo. environments.

TABLE 3 Oleic acid content and seed generation of soybean lines withdifferent combinations of mutant FAD2-1A and mutant FAD2-1B produced inthree environments. Oleic acid content (percent of total fatty acid)Population Columbia, FAD2-1A FAD2-1B Costa Rica¹ Portageville, MO² MO³

 

P137R 81.4 ± 5.7^(F5) 82.2 ± 1.2^(F7) 79.1 ± 1.3^(F8)

 

I143T 80.0 ± 4.0^(F5) 65.0 ± 4.3^(F7) 58.7 ± 7.7^(F8) 4 S117N P137R 81.1± 2.2^(F2) 81.7 ± 2.1^(F3) 77.3 ± 2.0^(F3) ¹Research station in CostaRica. Seeds of F5 generation of population of 2 and 3 were produced inwinter 2006-2007, while F2 seeds of population 4 were produced in winter2008-2009. ²Plants were grown in Delta Research Center, seeds of F7generation of the populations 2 and 3 were produced in summer 2008 andF3 generation of population 4 was produced in summer 2009. ³All of theplants were grown summer 2009 at the Bradford Research & ExtensionCenter, Columbia MO.

Table 4 illustrates that the high oleic acid phenotype is stable acrossmultiple growing environments, including Portageville, Mo., Columbia,Mo., Stoneville, Miss. and Knoxyille, Tenn. Soybean plants inheritingthe aabb genotype have oleic acid contents ranging from 72.3-83.2.

TABLE 4 Stability analysis of high oleic acid soybean lines across theenvironments Portageville, MO Columbia, MO Stoneville, MS Name MG 16:018:0 18:1 18:2 18:3 16:0 18:0 18:1 18:2 18:3 16:0 18:0 18:1 18:2 18:3S08-14692 (aabb) IV 7.7 3.9 80.8 3.7 4.0 8.7 3.5 78.8 4.9 5.6 8.4 3.877.7 6.8 3.3 S08-14709 (aabb) IV 6.6 2.9 80.1 5.0 5.4 6.8 3.0 74.3 9.06.9 7.3 3.2 80.9 4.7 3.9 S08-14705 (aabb) IV 6.9 2.6 83.2 3.8 3.5 6.53.1 80.5 4.7 5.2 7.6 3.3 78.3 7.7 3.1 S08-14700 (aabb) V 7.5 2.4 82.13.7 4.3 7.5 2.9 76.5 6.9 6.2 7.9 2.7 78.9 7.4 3.2 S08-14702 (aabb) V 6.63.3 83.2 2.8 4.1 7.0 3.4 72.3 10.6 6.7 7.1 3.4 80.7 5.7 3.2 S08-14717(aabb) V 7.8 2.7 81.8 3.8 4.0 7.8 3.2 76.4 6.6 5.9 8.0 2.6 80.1 6.3 3.0M23 (FAD2-1A parent) (aa) V 10.0 2.9 43.6 36.3 7.2 9.3 3.5 44.2 34.4 8.69.2 2.9 59.2 23.9 4.8 PI283327 (FAD2-1B parent) (bb) V 10.8 4.2 27.846.3 10.8 10.7 4.1 23.1 49.7 12.4 11.9 3.9 30.6 46.1 7.5 5002T (Check)(AABB) IV 11.2 4.3 23.8 53.1 7.6 11.2 4.2 19.8 55.1 9.6 11.3 4.5 23.953.8 6.5 Anand (Check) (AABB) V 12.6 3.1 19.4 55.6 9.4 12.0 3.4 18.255.4 11.0 12.6 3.3 20.1 55.6 8.5 N98-4445A (Check-high oleic) IV 8.9 3.855.8 29.0 2.6 9.3 4.3 46.7 36.2 3.5 9.0 3.1 63.8 21.9 2.2 Knoxville, TNName 16:0 18:0 18:1 18:2 18:3 18:1 Range Differences S08-14692 (aabb)8.0 3.4 80.1 4.1 4.3 80.8-77.7 3.1 S08-14709 (aabb) 6.9 2.9 81.1 4.0 5.081.1-74.3 6.8 S08-14705 (aabb) 7.1 2.9 80.7 5.7 3.7 83.2-78.3 4.9S08-14700 (aabb) 7.9 2.6 80.7 4.2 4.6 82.1-76.5 5.6 S08-14702 (aabb) 6.93.1 82.6 3.5 4.0 83.2-72.3 10.9 S08-14717 (aabb) 7.9 2.7 82.1 3.2 4.182.1-76.4 5.7 M23 (FAD2-1A parent) (aa) 9.5 2.8 52.0 29.7 6.1 59.2-43.615.6 PI283327 (FAD2-1B parent) (bb) 11.1 4.0 25.3 48.2 11.4 30.6-23.17.5 5002T (Check) (AABB) 11.7 4.1 21.7 54.9 7.6 23.9-19.8 4.1 Anand(Check) (AABB) 12.3 3.2 21.6 54.5 8.3 21.6-18.2 3.4 N98-4445A(Check-high oleic) 8.8 3.5 63.6 21.8 2.3 63.8-46.7 17.1

Lines S08-14692, S08-14709, S08-14705, S08-14700, S08-14702 andS08-14717 are soybean lines selected from a cross of lines M23×PI283327that inherit the mutant FAD2-1A alleles (aa) from M23 and the FAD2-1BP137R alleles (bb) from PI 283327 and are genotype aabb. Lines Anand and5002T are soybean lines that are wild-type for the FAD2-1A alleles (AA)and FAD2-1B alleles (BB) and have the genotype AABB. Line N98-4445A asoybean line that contains elevated oleic acid content and carries atleast six genes (QTLs) conditioning the high oleic phenotype.

Determination of Fatty Acid Content

Fatty acid profiles as a percent of total oil for each genotype withineach environment were determined by Gas Chromatography (GC) as describedby Oliva et al. (2006). In most cases, five individual seeds fromvarious strains and crosses were randomly selected for fatty acidanalysis. The fatty acid profiles as illustrated in FIG. 2, however,used between either 5 or 10 seeds for measurement. Each five or ten seedsample was placed in a paper envelope, and then manually crushed with ahammer. Oil was extracted by placing crushed seeds in 5 mLchloroform:hexane:methanol (8:5:2, v/v/v) overnight. Derivitization wasdone by transferring 100 μL of extract to vials and adding 75 μL ofmethylating reagent (0.25 M methanolic sodium methoxide:petroleumether:ethyl ether, 1:5:2 v/v/v). Hexane was added to bring samples toapproximately 1 mL. An Agilent (Palo Alto, Calif.) series 6890 capillarygas chromatograph fitted with a flame ionization detector (275° C.) wasused with an AT-Silar capillary column (Alltech Associates, Deerfield,Ill.). Standard fatty acid mixtures (Animal and Vegetable Oil ReferenceMixture 6, AOACS) were used as calibration reference standards.

As illustrated in FIGS. 2-4, “A” denotes a “wild-type” or non mutatedFAD2-1A allele such as carried by reference strain W 82. “a” denotes amutated FAD2-1A (mFAD2-1A) allele, such as carried by strain M23. “B”denotes a “wild-type” or non-mutated FAD2-1B allele. “b” denotes amutated FAD2-1B (mFAD2-1B) allele such as carried by strains PI 283327and PI 567189A. Thus “AA” denotes a homozygous FAD2-1A genotype, “aa”denotes a homozygous mFAD2-1A genotype, “BB” denotes a homozygousFAD2-1B genotype, “bb” denotes a homozygous mFAD2-1B genotype, Aadenotes a heterozygous FAD2-1A/mFAD2-1A genotype and Bb denotes aheterozygous FAD2-1B/mFAD2-1B genotype.

FIG. 2 is a bar graph showing the relative fatty acid content of fattyacid components 16:0, 18:0, 18:1, 18:2 and 18:3 in various allelicvariants of F7 progeny derived from M23×PI 283327 recombinant inbredlines (RILs). As can be seen in FIG. 2, progeny homozygous for wild-typeFAD2-1A and FAD2-1B (AABB) had oleic acid levels consistent with what isnormally found in nature i.e. around 20%. The corresponding byproduct ofoleic acid desaturation, linoleic acid levels were around 55%. Mutationsin FAD2-1B alone (AAbb) showed only a very minor increase in oleic acidcontent, ranging from between about 25% to about 30%. Remarkably,progeny with both the mFAD2-1A and mFAD2-1B (aabb) alleles had oleicacid levels around 80%, with the corresponding linoleic acid levelsbelow 5%.

As shown in FIG. 3, oleic acid content was further characterized andcompared to the parental lines M23 and PI 283327. Consistent with theresults in FIG. 2, seeds with wild-type alleles (AABB) had levels ofoleic acid around 20%. Seeds with genotypes of either the aaBB or AAbbhad levels of oleic acid around 40% or around 25% respectively. Asdemonstrated in FIG. 2, while mutations in FAD2-1B alone (AAbb) showedonly a very minor increase in oleic acid content, double mutant seedswith the mFAD2-1A and mFAD2-1B (aabb) alleles had oleic acid levels ofaround 80%. M23 and PI 283327 seeds had oleic acid levels of around 42%and 25%, respectively.

Similar to strain M23, 17D is a strain of soybean that has a mutation inthe FAD2-1A gene. As shown in FIG. 4, F2 seeds (produced in Costa Ricain early 2009) homozygous for this mutation showed a small increase inoleic acid levels from around 20% to around 25%. When strain 17D wascrossed with a line derived from PI 283327, F2 seeds containinghomozygous genes of both mFAD2-1A and mFAD2-1B (aabb) had an oleic acidcontent of around 80%. FIG. 4 also shows that various heterozygousgenotypes had varying levels of oleic acid illustrating that astratification of oleic acid levels may be obtained through a variationof FAD2-1A and FAD2-1B allele combinations. For example, heterozygousinheritance of 17D mFAD2-1A (Aa) and homozygous inheritance of mFAD2-1B(bb) resulted in seeds with around 45% oleic acid levels.

The initial investigation of both the FAD2-1 genotype and fatty acidphenotype in F2 seeds from Population 4 (FAD2-1A S117N×FAD2-1B P137cross) demonstrated the epistatic nature of the mutant alleles workingin combination, and the results revealed that only homozygouscombinations of both mutant FAD2-1A and FAD2-1B were capable ofproducing the high oleic acid phenotype. Of the 200 F2 seeds that werephenotyped, there were 12 individual F2 seeds with genotype FAD2-1 aabb,and they had an average oleic acid content of 81%, ranging from 75.2% to83.9% oleic acid (FIG. 4). The next highest oleic acid phenotype in theset was 48.8%, and that seed had the FAD2-1 Aabb genotype. For a tworecessive gene model, one sixteenth of the individuals should inheritthe phenotype; recovery of 12 individuals with the high oleic acidphenotype satisfies this expectation by Chi-Square test at the 0.05probability level.

Individuals with a single wild-type version of either FAD2-1A or FAD2-1Bin combination with three mutant FAD2-1 alleles (Aabb or aaBb) containedapproximately 40% oleic acid. No seeds from any of the other FAD2-1genotypes contained oleic acid levels above 49% of the seed oil.Individuals with two or more wild-type FAD2-1 alleles contained oleicacid content with a range of 18-47% of the seed oil.

The necessity of the homozygous FAD2-1A and FAD2-1B mutant combinationrequirement for the high oleic acid phenotype was confirmed in anindependent analysis of FAD2-1 genotype and fatty acid phenotype offield produced F2 seeds that contained homozygous FAD2-1A Δ alleles butwhich were segregating for FAD2-1B P137R alleles (Population 5). Whilethe average oleic acid level of those seeds with the aabb genotype was82.5%, aaBb seeds averaged 55.4%; aaBB seeds averaged 43.4% oleic acidin the seed oil. The presence of a single wild-type version of theFAD2-1B allele also prevented a high oleic acid content in the seed oil,although the magnitude of the difference was greater for the F2 seedsfrom Population 4.

Table 5 shows the relative oleic acid content for 14 soybean plant linesderived from M23×PI 283327 between 2006-2007 and 2007-2008. Asdesignated in Table 3, “MT” represents the maturity date in days afterAugust 1, i.e. an MT of 68 indicates that the line matured on October 8.Each of the 14 F6 lines were homozygous recessive for mFAD2-1A andmFAD2-1B. Furthermore, each of the 14 lines traced to a separate F2plant and are F2:6 recombinant inbred lines. These results derived fromseed grown in Costa Rica. Samples from 2006-2007 were of the F5generation, whereas samples derived from 2007-2008 were of the F6generation. Oleic acid concentrations were generally near to, or greaterthan 80%, ranging from around 79% to around 86%.

TABLE 5 Oleic acid content as percentage of total fatty acid for 14soybean plant lines derived from M23 × PI283327 grown in Costa Rica2006-07 2007-08 Line MT 08 18:1 (F5) 18:1 (F6) S08-14692 56 84.5 83.8S08-14693 60 84.1 75.8 S08-14700 68 84.5 84.5 S08-14701 68 82.0 85.5S08-14702 68 86.5 84.2 S08-14705 60 81.0 84.4 S08-14708 58 85.4 84.6S08-14709 60 83.2 82.4 S08-14711 65 83.9 82.7 S08-14715 68 79.6 82.2S08-14716 58 86.4 84.9 S08-14717 70 86.6 85.7 S08-14718 65 86.4 84.4S08-14719 85.0 83.4

Table 6 shows the fatty acid profiles for 14 soybean plant lines derivedfrom M23×PI 283327 performed in 2008. Each of the 14 F6 lines werehomozygous recessive for mFAD2-1A and mFAD2-1B. Furthermore, each of the14 lines traced to a separate F2 plant and is a F2:6 recombinant inbredline. Seed from the 14 soybean lines were grown in Portageville Mo.Oleic acid concentrations were generally near to, or greater than, 80%,and ranged from around 79% to around 85%.

TABLE 6 Fatty acid profiles for 14 F7 soybean plant lines derived fromM23 × PI 283327 grown in Portageville Missouri Range # of Line 16:0 18:018:1 18:2 18:3 (18:1) plants S08-14692 8.0 3.6 81.2 3.0 4.1 80.6-81.9 15S08-14693 8.5 3.2 79.3 4.6 4.5 77.7-80.7 3 S08-14700 8.1 3.2 82.0 2.74.2 80.7-83.9 15 S08-14701 7.7 3.4 83.0 2.4 3.4 81.9-84.5 15 S08-147027.0 3.8 82.9 2.4 3.9 81.5-84.4 15 S08-14705 8.3 3.9 82.7 1.7 3.481.5-83.9 6 S08-14708 7.6 3.9 82.3 2.1 4.2 80.2-83.8 9 S08-14709 7.6 3.581.3 3.0 4.6 76.4-82.2 15 S08-14711 8.4 4.2 80.8 2.4 4.2 79.0-81.6 15S08-14715 7.8 4.2 80.8 2.8 4.4 79.4-82.5 15 S08-14716 8.8 3.2 81.3 2.83.8 80.3-83.2 8 S08-14717 8.1 3.7 82.9 1.7 3.7 81.0-84.0 15 S08-147187.1 3.9 83.5 1.9 3.6 82.2-84.4 15 S08-14719 8.7 2.8 81.6 3.5 4.079.3-83.6 22 M23 parent 10.2 3.3 43.8 35.9 6.8 PI 283327 11.0 4.1 26.547.8 10.6 parent

Table 7 shows the fatty acid profiles from analyses in 2008 for 12 F2soybean plant lines derived from 17D×S08-14788 (Jake×PI 283327). Oleicacid levels ranged from about 75% to about 84%.

TABLE 7 Fatty acid profiles for 12 F2 soybean lines derived from 17D ×S08-14788(Jake × PI283327) Line 16:0 18:0 18:1 18:2 18:3 10 7.0 2.7 83.92.4 4.1 41 8.4 3.0 75.2 7.6 5.8 43 7.9 3.2 81.2 2.9 4.8 46 7.5 2.8 83.02.4 4.4 67 7.6 3.2 81.5 2.6 5.0 92 7.4 3.4 81.4 2.8 4.9 98 7.5 3.0 82.62.5 4.4 104 8.3 3.2 81.1 2.8 4.6 106 7.5 2.8 80.9 3.1 5.7 129 7.4 3.382.3 2.9 4.2 159 8.9 3.0 79.5 2.8 5.7 197 7.9 3.1 80.6 3.5 4.8

Seed (grown in Portageville, Mo. in 2008) derived from a cross betweenM23 and PI 567189A (M23×PI 567189A) were also analyzed to determinerelative amounts of oleic acid. FIG. 5 represents genotype and phenotypeanalysis for plants that inherited either a wild-type (AA) or deletedversion (aa) of the FAD2-1A gene and either a wild-type (BB) or theI143T mutant allele (bb) of FAD2-1B from PI 567189A that differs fromthe in FAD2-1B allele present in PI 283327 (described above). As shownin FIG. 5, the PI 567189A allele was “weaker” than the PI 283327 alleleof mFAD2-1B. Whereas soybean plants inheriting homozygous alleles ofboth PI 283327 and M23 consistently had levels of oleic acid around 80%,soybean plants inheriting homozygous mutant FAD2-1A and FAD2-1B allelesfrom PI 567189A and M23 had oleic acid content around 65%.

Seed derived from a cross between Jake and PI 283327 (Jake×PI 283327)were also analyzed to determine their fatty acid profile. FIG. 6represents genotype and phenotype analysis for plants that inheritedeither a wild-type (AA) version of the FAD2-1A gene and either awild-type (BB) or the P137R mutant allele (bb) of FAD2-1B from PI 283327that differs from the mFAD2-1B allele present in PI 567189A (describedabove). As shown in FIG. 6, the PI 283327 mFAD2-1B allele on thewild-type Jake background (AAbb) had modest effects on oleic acidlevels. Whereas, seeds inheriting the AABB genotypes had oleic acidlevels of around 20%, seeds inheriting the AAbb genotypes had only aslight increase in oleic acid levels to around 28%.

Taken together these data indicate that plants inheriting loss offunction or reduced activity mutations in both the FAD2-1A gene and theFAD2-1B gene produced seed with high levels of oleic acid contentranging from about 75% to about 85%.

The full fatty acid profiles of the seeds of contrasting FAD2-genotypicclasses produced from Populations 2, 3, and 4 in this study revealedadditional alterations in palmitic acid, linoleic acid, and linolenicacid content (Table 6). As expected for a major decrease in seedexpressed FAD2 enzyme activity that results in an accumulation of oleicacid, the FAD2 reaction products linoleic acid and linolenic acid weredramatically reduced in the high oleic FAD2-1A and FAD2-1B homozygousmutant lines when either of the FAD2-1A mutations were present alongwith the FAD2-1B P137R or I143T alleles.

Table 8 shows fatty acid profiles for different homozygous FAD2-1genotypes in four segregating populations developed by crossing soybeanlines carrying different sources of mutant FAD2-1A alleles withdifferent sources of mutant FAD2-1B alleles.

TABLE 8 Fatty acid profiles of various genotypes. Fatty Acid 16:0 18:018:1 18:2 18:3 Population 1 (Jake1 × PI 283327) BB 12.2 ± 0.9 3.9 ± 0.520.5 ± 2.6 53.4 ± 2.8 10.0 ± 0.3  (n = 24) bb 11.2 ± 0.7 3.8 ± 0.6 29.4± 6.0 47.0 ± 5.1 8.7 ± 0.5 (n = 30) Population 2 (M23 × PI283327) AABB12.3 ± 0.5 3.7 ± 0.4 19.9 ± 3.3 55.4 ± 2.7 8.7 ± 1.0 (n = 5) AAbb 11.0 ±0.5 3.9 ± 0.4 30.8 ± 5.2 45.9 ± 4.6 8.5 ± 0.9 (n = 5) aaBB 10.8 ± 0.83.8 ± 0.6 39.4 ± 5.7 37.1 ± 4.8 8.9 ± 1.2 (n = 14) aabb  7.9 ± 0.7 3.7 ±0.6 82.2 ± 1.2  2.3 ± 0.6 3.9 ± 0.5 (n = 16) Population 3 (M23 × PI567189A) AABB 12.5 ± 0.9 2.9 ± 0.4 26.3 ± 7.4 51.4 ± 6.4 6.1 ± 1.2 (n =11) AAbb 12.4 ± 0.8 2.8 ± 0.4 31.1 ± 4.5 47.5 ± 3.3 6.1 ± 1.0 (n = 3)aaBB 10.3 ± 0.6 2.8 ± 0.3 48.2 ± 7.2 32.5 ± 6.1 6.2 ± 0.9 (n = 1) aabb 8.4 ± 0.8 2.6 ± 0.4 80.0 ± 4.0  5.0 ± 3.0 3.8 ± 0.6 (n = 16) Population4 F2 (17D × S08-14788) AABB 12.3 ± 0.9 3.2 ± 0.3 20.1 ± 0.9 55.7 ± 1.08.7 ± 0.6 (n = 5) AAbb 12.1 ± 1.0 3.4 ± 0.5 26.5 ± 4.5 47.8 ± 3.7 10.2 ±0.9  (n = 5) aaBB 11.7 ± 0.3 3.0 ± 0.2 26.8 ± 1.4 48.2 ± 0.7 9.9 ± 0.5(n = 6) aabb  7.8 ± 0.5 3.1 ± 0.2 81.1 ± 2.2  3.2 ± 1.4 4.9 ± 0.6 (n =12) Population 4 F 2:3 (17D × S0814788) AABB  9.6 ± 0.6 3.9 ± 0.4 22.4 ±2.9 56.0 ± 2.8 8.2 ± 0.9 (n = 5) AAbb 10.5 ± 0.5 3.8 ± 0.3 23.1 ± 2.554.0 ± 2.6 8.6 ± 0.5 (n = 4) aaBB  9.3 ± 0.6 3.2 ± 0.3 35.0 ± 7.8 42.9 ±5.9 9.6 ± 2.2 (n = 6) aabb  6.9 ± 0.4 3.2 ± 0.2 77.3 ± 2.0  6.3 ± 1.56.3 ± 0.6 (n = 11) *AA = wild-type FAD2-1A alleles, aa = mutant FAD2-1Aalleles derived from M23 or17D, BB = wild-type FAD2-1B alleles, bb =mutant FAD2-1B alleles derived from PI 283327 or PI 567189A.

By evaluating the proportions of oleic, linoleic, and linolenic acidspresent in the oil extracted from mature seeds, the relative FAD2 andFAD3 desaturase activities of the developing seeds were determined forthe contrasting homozygous FAD2-1 genotypes from each population. TheFAD2-1 AABB genotypes contained FAD2 desaturase activities (final oleicacid content divided by the sum of final oleic, linoleic, and linolenicacid contents) of 76%, 76%, and 74% for Population 2, Population 3, andPopulation 4, respectively. The FAD2-1 aabb genotypes contained FAD2desaturase activities of 7%, 10%, and 14%, for Population 2, Population3, and Population 4, respectively. Also noted is that the accumulationof linolenic acid follows a different pattern for the FAD2-1 aabb mutantlines compared to the FAD2-1 AABB lines, with increased FAD3 desaturaseactivity (final linolenic acid content divided by the sum of finallinoleic and linolenic acid contents) for the FAD2-1 mutant lines.

While no significant differences were observed for the stearic acidlevels in the contrasting FAD2-1 genotypes, the aabb mutant linesconsistently produced lower palmitic acid levels than lines with theAABB genotype. The most dramatic change was for Population 2. In thatcase, the content of palmitic acid was 7.9% for the aabb mutant linescompared to 12.3% for the AABB lines.

Because of the concern that improvement in fatty acid profiles mighthave negative impacts on the total oil and protein profiles of theseeds, we also evaluated the protein and oil contents for the fieldproduced F2:3 seeds from Population 4. There were no significantdifferences in the protein or oil contents among the differenthomozygous FAD2 genotypes, or with those lines compared to eitherWilliams 82 or the 17D parental line. The FAD2-1B P137R allele donorparental line had a minor decrease in the average oil content and thehighest mean protein content of all of the lines examined.

Genotyping High Oleic Acid Content Soybean Lines PI 283327 and PI567189A FAD2-1B Alleles from Wild-Type FAD2-1B Alleles

Genotyping assays were designed to distinguish the PI 283327 and PI567189A FAD2-1B alleles from wild-type alleles. The genotyping assayswork by asymmetric gene-specific real-time PCR amplification of genomicDNA in the FAD2-1B region surrounding the c410g and t428c singlenucleotide polymorphisms (SNPs) in the presence of a fluorescentlylabeled SimpleProbe (Roche Applied Sciences). After amplification, thePCR products are subjected to a melting curve analysis which tracks thedissociation kinetics of the SimpleProbe from the target DNA. TheSimpleProbe has a characteristic melting profile for homozygouswild-type, heterozygous, and homozygous mutant alleles.

The SimpleProbe, GmFAD2-1B, was designed to detect wild-type,heterozygous, and homozygous mutant alleles. GmFAD2-1B SimpleProbeconsists of 5′-SPC (simple probechemistry)-AGTCCCTTATTTCTCATGGAAAATAAGC-Phosphate-3′ (SEQ ID NO 11). TheC to G mutation and T to C mutation are indicated by underline.Genotyping reactions were performed with a 5:2 asymmetric mix of primers(5′-ACTGCATCGAATAATACAAGCC-3′ (SEQ ID NO: 12); at 2 μM finalconcentration, and 5′-TGATATTGTCCCGTCCAGC-3′(SEQ ID NO: 13); at 5 μMfinal concentration). Reactions were carried out in 20 μl; containingtemplate, primers, 0.2 μM final concentration of SimpleProbe, and 0.2×Titanium Tag polymerase (BD Biosciences, Palo Alto, Calif.). Genotypingreactions were performed using a Lightcycler 480 II real time PCRinstrument (Roche), using the following PCR parameters: 95° C. for 5minutes followed by 40 cycles of 95° C. for 20 seconds, 60° C. for 20seconds, 72° C. for 20 seconds, and then a melting curve from 55° C. to70° C. When DNA from PI 283327 and PI 567189A is amplified with genespecific primers and used in melting curve analysis with theSimpleProbe, a mismatch between the Simpleprobe and the amplicon resultsin altered disassociation kinetics. Each genotype produced acharacteristic melting profile, as measured by Tm of the negative firstderivative of the disappearance of fluorescent signal. PI 283327 and allsoybean lines with similar FAD2-1B genotype have a characteristic peakof 56.7° C., while PI 567189A yielded a characteristic peak at 60.2° C.M23 and Jake (wild-type for FAD2-1B) have a peak at 62.5° C.Heterozygous individual's genotype showed two peaks at either 56.7° C.or 60.2° C. and 62.5° C.

Genotyping for three populations Jake×PI 283327, M23×PI 283327, M23×PI567189A, were performed with SimpleProbe assay as described. FIG. 7graphically represents a melting curve analysis with peaks correspondingto homozygous Mutant (bb), wild-type (BB), and Heterozygous (Bb) allelesof FAD2-1B and mFAD2-1B genes.

Effect of Temperature on Oleic Acid Content

Although there is evidence of influence of temperature on the soybeanseed oleic acid content, two of our three high oleic acid soybeangenotypes proved to be capable of producing a high and stable oleic acidcontent in three environments. Moreover, there was no reduction in oiland protein content in the evaluated high oleic acid soybean lines.Soybean lines with the combination of FAD2-1A Δ and FAD2-1B I143Talleles from population 3 failed to produce the high oleic acidphenotype when grown in the nontropical environments. A possibleexplanation is the mutation in the FAD2-1B allele of PI 567189 A encodesat least nominal enzyme function. This explanation is supported by thefact that the I143T substitution is in a less conserved amino acid ofthe FAD2 enzyme than the P137R substitution. Other than that, the higholeic acid soybean lines showed a reduction of 4% at most when they weregrown in the cooler environment, with a small variation in the oleicacid content. It will be necessary to test the performance of these higholeic acid soybean lines in the main North American soybean growinglocations in more northern latitudes. The mutant FAD2-1A and FAD2-1Balleles will have to be combined in soybean lines with the appropriatematurity for those experiments to be conducted. However, based on thestability of the trait that we have observed so far, any reduction ofoleic acid content due to the environment is likely to be minor becausevery little FAD2 enzyme activity remains in developing seeds in themutant FAD2-1A and FAD2-1B lines. An additional factor is that the enduse market has not matured sufficiently to define the exact oleic acidcontent desired for different oil uses. Another question that should beaddressed is whether the trait will affect yield or other agronomictraits. It has been reported that the transgenic soybean lines with theFAD2-1 genes being silenced did not show any yield drag or abnormalphysiology characteristics.

The methods and strains, outlined above, function to produceconventional soybean varieties containing an enhanced nutritional oilprofile trait high in oleic acid oil. The current yearly demand or oleicacid is approximately four million tons of high oleic acid oil andgrowing. This figure translates to an annual production of two millionacres of high oleic acid soybean to meet the current demand. Theavailability of soybeans with enhanced oil profile traits may influencethe market and increase demand, particularly if the domestic biofuelcapacity increases.

As outlined above, transgenic technology is not required, thuseliminating the need for the expensive and time consuming regulatoryprocess. The developed perfect molecular markers and soybean germplasmprovide an efficient way to rapidly integrate these desirable traitsinto additional commercial soybean lines.

Industry has not had access to non-transgenic elite soybean varietieswith the high oleic acid trait. The high oleic acid soybean oil islikely to provide a replacement in the food industry for foodformulations that previously used partially hydrogenated vegetable oil.Currently, low linolenic acid soybean oil can fulfill some of the demandfor alternatives to the trans fat-containing partially hydrogenatedvegetable oil. High oleic acid soybean oil adds value by improvingfunctionality of soybean oil in many products such as improving coldflow of biodiesel; better lubricants to withstand high temperature andwider use in foods, pharmaceuticals and other products.

Example 2 Generation of High Oleic Acid Content Soybean Seeds UsingStandard Breeder Grower Methods

Soybean plant strains are analyzed for mutations that result in loss offunction or reduced biological activity of the FAD2-1A or FAD2-1B genesas described above. Soybean plant lines exhibiting impaired activity ineither FAD2-1A or FAD2-1B as measured by oleic acid content phenotype,are crossed (mFAD2-1A×mFAD2-1B) to generate progeny that carry both aFAD2-1A mutation a FAD2-1B mutation. These mutations are stablyinherited and function synergistically to produce seed with high levelsof oleic acid. Fatty acid compositions are analyzed from seed of soybeanlines derived from the parental cross using gas chromatography. Seed ofthe transformed plants exhibit high levels of oleic acid between about65% to about 85%.

Example 3 Selection of High Oleic Acid Soybean Lines with AdditionalDesirable Traits

In certain embodiments it may be desirable to select soybeans plantswith seeds having high oleic acid content as well as additionaldesirable traits with various phenotypes of agronomic interest. Examplesof additional desirable traits may be, but not limited to, diseaseresistance, pest resistance, pesticide resistance, accelerated growthrate, high seed yield, ability to grow in diverse environments etc.

A soybean plant with loss of function or reduced activity mutations inFAD2-1A and FAD2-1B is crossed with a soybean plant with one or moredesirable traits. Progeny from the cross are analyzed for the presenceof the desirable genotypic and phenotypic characteristics deriving fromFAD2-1A/FAD2-1B double mutants and the soybean plants with additionaldesirable traits.

Example 4 Generation of Dominant Negative FAD2 Transgenic Plants

A soybean nucleotide sequence with at least 80%, 90%, 95%, 98%, or 99%sequence identity to the sequence of SEQ ID NO: 1 and or SEQ ID NO: 2and or SEQ ID NO: 3 and or SEQ ID NO: 4 is cloned into an expressionvector. The resulting expression constructs are used for transformationof soybean using biolistic methods described below.

The expression vector may have a promoter that functions to express adominant negative form of mFAD2-1B at levels greater than those seenwhen expressed with the endogenous or wild-type promoter.

Linear DNA fragments containing the expression constructs for thedominant negative expression of mFAD2-1B desaturase genes are stablyintroduced into soybean (Asgrow variety A3244 or A4922A32) by theparticle bombardment method of McCabe et al. (1988), Bio/Technology,6:923-926 or via cocultivation with Agrobacterium tumefaciens, strainABI. (Martinell, U.S. Pat. No. 6,384,310). Transformed soybean plantsare identified by the genotyping assays described above.

Fatty acid compositions are analyzed from seed of soybean linestransformed with the dominant negative expression constructs using gaschromatography.

Example 5 Generation of High Oleic Acid Content Soybean Seeds

Soybean plant seeds are analyzed for spontaneous mutations that resultin elevated oleic acid phenotypes, as described above. Soybean plantlines exhibiting impaired activity in either FAD2-1A or FAD2-1B asmeasured by oleic acid content phenotype, are crossed (i.e.mFAD2-1A×mFAD2-1B) to generate progeny that carry both a FAD2-1Amutation a FAD2-1B mutation. These mutations are stably inherited andfunction synergistically to produce seed with high levels of oleic acid.Fatty acid compositions are analyzed from seed of soybean lines derivedfrom the parental cross using gas chromatography. Seed of thetransformed plants exhibit high levels of oleic acid (over 80%).

The description of the specific embodiments reveals general conceptsthat others can modify and/or adapt for various applications or usesthat do not depart from the general concepts. Therefore, suchadaptations and modifications should and are intended to be comprehendedwithin the meaning and range of equivalents of the disclosedembodiments. It is to be understood that the phraseology or terminologyemployed herein is for the purpose of description and not limitation.Certain terms with capital or small letters, in singular or in pluralforms, may be used interchangeably in this disclosure.

All references mentioned in this application are incorporated byreference to the same extent as though fully replicated herein.

1. A method of selecting soybean plants with seed having an oleic acid content of between about 65% to about 85%, said method comprising: crossing a first soybean plant having one or more mutations in a first polynucleotide sequence encoding a FAD2-1A gene SEQ ID NO: 5 with a second soybean plant having one or more mutations in a second polynucleotide sequence encoding a FAD2-1B gene SEQ ID NO:
 6. 2. The method of claim 1 wherein the first polynucleotide sequence further comprising having at least 95% identity assessed as sequence homology with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:
 4. 3. The method of claim 1 wherein the second polynucleotide sequence further comprising having at least 95% identity assessed as sequence homology with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:
 2. 4. The method of claim 1 wherein the one or more mutations interfere with a function of a protein encoded by the first polynucleotide sequence encoding FAD2-1A and a protein encoded by the second polynucleotide sequence encoding FAD2-1B.
 5. The method of claim 1 wherein the one or more mutations result in a loss of function and/or a reduced activity of a protein encoded by the first polynucleotide sequence and a protein encoded by the second polynucleotide sequence.
 6. The method of claim 1, wherein a mutation in the second polynucleotide sequence encoding a FAD2-1B polypeptide includes at least one mutation selected from the group consisting of: a. a non-conserved amino acid substitution at amino acid position 137 b. a non-conserved amino acid substitution at amino acid position 143
 7. The method of claim 6 wherein the at least one mutation in the polypeptide comprises a polar amino acid.
 8. The method of claim 7 where the polar amino acid is selected from the group consisting of: arginine, glycine, serine, threonine, cysteine, asparagine, tyrosine, glutamine, lysine and histidine.
 9. The method of claim 1 wherein the mutation in the first polynucleotide sequence encoding a FAD2-1A polypeptide includes at least one mutation comprising at least one non-conserved amino acid substitution at amino acid position
 117. 10. The method of claim 9 wherein the soybean plant contains at least one non-conserved amino acid substitution as asparagine.
 11. The method of claim 1 further comprising, predicting one or more amino acid substitutions that result in a loss of function or a reduced activity of the protein encoded by the first polynucleotide sequence and the second polynucleotide sequence before crossing the first and second soybean plants.
 12. A stably reproducing line of soybean plants produced according to the method of claim 1, wherein the stability of progeny is assessed by a population of progeny including at least 2500 seeds present an oleic acid content of between about 65% to about 85% determined as a percentage of total fatty acid content.
 13. The method of claim 1 further comprising crossing the progeny to produce a recombinant inbred line.
 14. A recombinant inbred soybean line produced according to the method of claim
 13. 15. A stably reproducing population of soybean seeds having one or more mutations in a first and a second polynucleotide sequence wherein the first polynucleotide sequence encodes for expression of FAD2-1A SEQ ID NO: 5 and the second polynucleotide sequence encodes for expression FAD2-1B SEQ ID NO: 6, wherein seed from said plant has about 65% to about 85% oleic acid content.
 16. The soybean seeds of claim 15 wherein the first polynucleotide sequence of has at least 95% identity determined as sequence homology with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:
 4. 17. The soybean seeds of claim 15 wherein the second polynucleotide sequence of has at least 95% identity determined as sequence homology with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:
 2. 18. The soybean seeds of claim 15 wherein the one or more mutations interfere with a function of a protein encoded by the first polynucleotide sequence encoding FAD2-1A and the second polynucleotide sequence encoding FAD2-1B.
 19. The soybean seeds of claim 15 wherein the one or more mutations result in a loss of function and/or reduced activity of a protein encoded by the first polynucleotide sequence and the second polynucleotide sequence.
 20. The soybean seeds of claim 15, wherein a mutation in the second polynucleotide sequence encoding a FAD2-1B polypeptide includes at least one mutation selected from the group consisting of: a. a non-conserved amino acid substitution at amino acid position 137 b. a non-conserved amino acid substitution at amino acid position 143
 21. The soybean seeds of claim 20 wherein the at least one mutation in the polypeptide comprises a polar amino acid.
 22. The soybean seeds of claim 21 wherein the polar amino acid is selected from the group consisting of: arginine, glycine, serine, threonine, cysteine, asparagine, tyrosine, glutamine, lysine and histidine.
 23. The soybean seeds of claim 15 wherein the mutation in the first polynucleotide sequence encoding a FAD2-1A polypeptide includes at least one mutation comprising at least one non-conserved amino acid substitution at amino acid position
 117. 24. The soybean seeds of claim 23 wherein the at least one non-conserved amino acid substitution is asparagine.
 25. The soybean seeds of claim 15 wherein one or more amino acid substitutions that result in a loss of function or a reduced activity of the protein encoded by the first polynucleotide sequence and the second polynucleotide sequence were predicted.
 26. The soybean seeds of claim 15, wherein progeny, derived from a cross of said soybean plant, and a second soybean plant, stably inherit a trait, said trait comprising seed having an oleic acid content of between about 65% to about 85%.
 27. The soybean plant of claim 26 further comprising crossing the progeny to produce a recombinant inbred line. 